The most commonly used agents in the treatment of acute asthma are drugs that activate human beta2-adrenergic receptors (beta2ARs). Following treatment with agonist, signaling by beta2ARs is greatly reduced by acute and chronic desensitization processes involving several distinct, but overlapping mechanisms - rapid phosphorylation by G- protein coupled receptor kinases (GRKs) and protein kinases A and C, receptor endocytosis into endosomes, and downregulation. Downregulation, which results in a decline in total cellular receptors and lowered sensitivity to beta-agonists, occurs with prolonged exposures to agonist and involves the sorting of receptors to multiple cellular destinations. Our long-range research goal is to understand the mechanisms of endosome sorting of beta2ARs and how these sorting steps determine receptor levels. The objective of the research proposed in this application is to define the role of receptor phosphorylation as a determinant of the intracellular targeting of beta2ARs to the cell surface, to the pericentriolar recycling compartment (PCRC), and to lysosomes. The central hypothesis is that sorting of beta2ARs to the recycling or degradative pathway is regulated by distinct phosphorylation steps and that these steps are key determinants of beta2AR levels. The rationale for the proposed studies is that once the regulation of beta2AR targeting is understood, therapeutic strategies can be designed to alter these pathways and increase the level of receptors available for activation. We are uniquely qualified to successfully complete this research given our expertise in the fields of endosome trafficking and receptor structure/function biology and because of the active collaboration amongst the investigators. We expect to test our central hypothesis and achieve the objective of this proposal by pursuing the following three specific aims: 1) Determine the effect of beta2AR traffic through the PCRC during chronic exposures to agonist and the role of this pathway in the efficient resensitization of receptors following downregulation, 2) Establish GRK2 and GRK5 as regulators of beta2AR sorting to the recycling or degradative pathways during chronic exposures to agonist, and 3) Identify distinct phosphorylation sites within the cytoplasmic tail of the beta2AR that target receptors to the recycling or degradative pathways. In this proposal, we will employ innovative approaches such as inducible expression systems to predictably manipulate the levels of rab11 and GRK and restoration microscopy to define beta2AR subcellular localization with high resolution. Further, we will determine the role in chronic desensitization of specific serine residues in the receptor's cytoplasmic tail that we have recently identified as being essential for acute receptor desensitization. We expect to fully define the role of phosphorylation in determining the intracellular destinations of beta2ARs and to identify how sorting to the PCRC effects receptor function. These results will be significant because they are expected to provide new targets for therapeutic interventions to increase beta2AR levels and thereby improve beta-agonist responsiveness in patients suffering from acute asthma.